Since the sixties of the last century, it has been proposed to use the specific binding power of the immune system (T cells and antibodies) to selectively kill tumor cells but leave alone the normal cells in a patient's body. Many tumor antigens that could be targeted by, in particular, antibodies, like carcino-embryonic antigen (CEA), alpha-fetoprotein (AFP) and so on, have been suggested since those days, but for essentially all of these antigens, expression is associated with normal tissue as well. Thus, so far, selective killing of aberrant cells has been an elusive goal.
The primary immunological function of MHC molecules is to bind and to “present” antigenic peptides to form an MHC-peptide (MHC-p) complex on the surface of cells for recognition and binding by antigen-specific T-cell receptors (TCRs) of lymphocytes. Antigenic peptides are also referred to as epitopes, both of which have basically the same meaning throughout the application. Two classes of MHC-p complexes can be distinguished with regard to their function:
(i) MHC class I-p complexes can be expressed by almost all nucleated cells in order to attract CD8+ cytotoxic T cells, and
(ii) MHC class II-p complexes are constitutively expressed only on so-called antigen-presenting cells (APCs), such as B lymphocytes, macrophages or dendritic cells (DCs).
MHC class I-p complexes are composed of a variable heavy chain, an invariable β-microglobulin and an antigenic peptide. The MHC class II molecules are characterized by distinctive α and β polypeptide subunits that combine to form αβ heterodimers characteristic of mature MHC class II molecules. Differential structural properties of MHC class I and class II molecules account for their respective roles in activating different populations of T lymphocytes. Cytotoxic TC lymphocytes (CTLs) bind antigenic peptides presented by MHC class I molecules. Helper TH lymphocytes bind antigenic peptides presented by MHC class II molecules. MHC class I and class II molecules differentially bind CD8 and CD4 cell adhesion molecules. MHC class I molecules are specifically bound by CD8 molecules expressed on CTLs, whereas MHC class II molecules are specifically bound by CD4 molecules expressed on helper TH lymphocytes.
The sizes of the antigenic peptide-binding pockets of MHC class I and class II molecules differ; class I molecules bind smaller antigenic peptides, typically eight to ten amino acid residues in length, whereas class II molecules bind larger antigenic peptides, typically 13 to 18 amino acid residues in length.
In humans, MHC molecules are termed human leukocyte antigens (HLA). HLA-associated peptides are short, encompassing typically 9 to 25 amino acid residues. Humans synthesize three different types of class I molecules designated HLA-A, HLA-B, and HLA-C. Human class II molecules are designated HLA-D, e.g., HLA-DR.
The MHC expressed on all nucleated cells of humans and of animals plays a crucial role in immunological defense against pathogens and cancer. The transformation of normal cells to aberrant cancer cells involves several major changes in gene expression. This results in profound changes in the antigenic composition of cells. It is well established that new antigenic entities are presented as MHC-restricted tumor antigens. As such, the MHC class I and MHC class II systems may be seen as nature's proteomic scanning chips, continuously processing intracellular proteins, generating antigenic peptides for presentation on the cell surface. If these antigenic peptides elicit an immune reactivity, the transformed cells are killed by the cellular immune system. However, if the transformed cells resist immune-mediated cell killing, cancer may develop.
Antibodies that bind MHC class I molecules on various cell types have been studied in detail for their mode of action. Mouse monoclonal antibodies that bind the MHC class I α1 domain of the MHC class I α chain induce apoptosis in activated T cells, but not in resting T cells. Other reports mention antibodies specific for, e.g., the α3 domain of MHC class I, which induce growth inhibition and apoptosis in B-cell-derived cancer cells. However, in this case, a secondary cross-linking antibody was required for the induction of apoptosis (A. E. Pedersen et al., Exp. Cell Res. 1999, 251:128-34).
Antibodies binding to β2-microglobulin (β2-M), an essential component of the MHC class I molecules, also induce apoptosis. Several hematologic cancer cells treated with anti-132M antibodies were killed efficiently, both in vitro and in vivo (Y. Cao et al., Br. J. Haematol. 2011, 154:111-121).
Thus, it is known that binding of MHC class I or MHC class II molecules by several anti-MHC antibodies can have an apoptosis-inducing effect. However, the therapeutic application of these anti-MHC antibodies has been hampered by the lack of target cell specificity. Since these antibodies are directed primarily against a constant domain of the MHC molecule, the cell surface expression of the MHC constant domain determines whether or not a cell can be triggered by the antibody to undergo apoptosis. Because MHC class I and MHC class II molecules are expressed on both normal and aberrant cells, it is clear that these antibodies cannot discriminate between normal and aberrant cells. As a consequence, their therapeutic value is significantly reduced, if not abolished by the side effects caused by unwanted apoptosis of healthy cells. According to the invention, antibodies that specifically recognize MHC-presented antigenic peptides derived from cancer antigens would, therefore, dramatically expand the therapeutic repertoire, if they could be shown to have anti-cancer cell activity. In addition, current methods to induce apoptosis via MHC class I or MHC class II may depend on external cross-linking of anti-MHC antibodies.
Obtaining antibodies binding to MHC-p complexes and not binding to MHC molecules not loaded with the antigenic peptide remains a laborious task and several failures have been reported. The first available antibodies have been obtained after immunization of mice with recombinant MHC-p complexes or peptide-loaded TAP-deficient antigen-presenting cells. More recently, antibodies have been obtained by selection from phage-antibody libraries made from immunized transgenic mice or by selection from completely human antibody phage libraries. Immunization with MHC-p complexes is extremely time consuming. Moreover, antibodies of murine origin cannot be used repetitively in patients because of the likely development of a human anti-mouse antibody response (so-called anti-drug antibodies, ADA). Antibodies derived from phage display, in general, display low affinity for the antigen and thus may require additional modifications before they can be used efficiently. According to the invention, the antibody specificities are preferably selected through phage (or yeast) display, whereby an MHC molecule loaded with a cancer-related peptide is presented to the library. Details are given in the experimental part. The antibody specificities according to the invention are checked for specificity to the MHC-peptide complex and should not recognize (to any significant extent) MHC loaded with irrelevant peptides or the peptides by themselves.
Cancer is caused by oncogenic transformation in aberrant cells, which drives uncontrolled cell proliferation, leading to misalignment of cell-cycle checkpoints, DNA damage and metabolic stress. These aberrations should direct tumor cells toward an apoptotic path that has evolved in multi-cellular animals as a means of eliminating abnormal cells that pose a threat to the organism. Indeed, most transformed cells or tumorigenic cells are killed by apoptosis. However, occasionally, a cell with additional mutations that enable avoidance of apoptotic death survives, thus enabling its malignant progression. Thus, cancer cells can grow, not only due to imbalances in proliferation and/or cell cycle regulation, but also due to imbalances in their apoptosis machinery. Imbalances like, for example, genomic mutations resulting in non-functional apoptosis-inducing proteins or over-expression of apoptosis-inhibiting proteins, form the basis of tumor formation. Fortunately, even cells that manage to escape the apoptosis signals this way when activated by their aberrant phenotype, are still primed for eradication from the organism. Apoptosis in these aberrant cells can still be triggered upon silencing or overcoming the apoptosis-inhibiting signals induced by mutations. Traditional cancer therapies can activate apoptosis, but they do so indirectly and often encounter tumor resistance. Direct and selective targeting of key components of the apoptosis machinery in these aberrant cells is a promising strategy for development of new anti-tumor therapeutics. Selective activation of the apoptosis pathway would allow for halting tumor growth and would allow for induction of tumor regression.
A disadvantage of many, if not all, anti-tumor drugs currently on the market or in development, which are based on targeting the apoptosis machinery, is that these drugs do not discriminate between aberrant cells and healthy cells. This non-specificity bears a challenging risk for drug-induced adverse events. Examples of such unwanted side effects are well known to the field: radiotherapy and chemotherapeutics induce apoptosis only as a secondary effect of the damage they cause to vital cellular components. Not only aberrant cells are targeted, though, in fact, most proliferating cells including healthy cells respond to the apoptosis-stimulating therapy. Therefore, a disadvantage of current apoptosis-inducing compounds is their non-selective nature, which reduces their potential.
In an earlier application (WO2007/073147; Apoptosis-inducing protein complexes and therapeutic use thereof, incorporated herein by reference), it is disclosed that a polypeptide complex achieves the goal of (specifically) killing, e.g., tumor cells by specifically targeting these cells and, as a result, induces apoptosis in these tumor cells. Although it is undesirable to be bound by theory, at present, it is believed that this is the result of cross-linking of cell-surface-expressed protein-protein complexes by multiple interactions with the multivalent polypeptide complex of that invention.
Two interlinked signaling pathways control apoptosis activation. Intracellular signals, such as DNA damage, drive apoptosis primarily through the intrinsic pathway, controlled by the Bcl-2 protein family. Extracellular signals, usually generated by cytotoxic cells of the immune system such as natural killer cells or cytotoxic T cells, trigger apoptosis mainly through the extrinsic pathway. Both pathways stimulate caspases with apoptosis-inducing activity. Caspases are a family of cysteine proteases, which are present in most cells as pro-caspases and which are activated through the so-called caspase cascade. Apoptotic signals first stimulate upstream initiator caspases (amongst others, caspases 8, 9 and 10) by recruiting them into specific signaling complexes that promote their multimerization. In turn, these caspases in signaling complexes activate downstream effector caspases (including caspases 3, 6 and 7) by proteolytic processing. These effector caspases then, in turn, process various cellular proteins, resulting in the apoptotic cell death program.
Some viruses (or at least some of their proteins), such as chicken anemia virus (CAV), parvovirus minute virus of mice (MVM), engineered herpes simplex virus, reovirus, vesicular stomatitis virus, adenovirus type 2 and poxvirus such as vaccinia, can selectively and preferentially kill tumor cells. These viruses do so through activation of the apoptosis machinery of the aberrant cell infected by the virus. The viruses are able to specifically provide the effective apoptosis-inducing death signal, which can interact with one or more of the derailed cancer processes. Fortunately, these viruses (or their proteins) have the ability to efficiently target cell death program in aberrant cells, although this cell death program might be derailed as a consequence of its aberrant nature. Two oncolytic virus-based therapies are tested in clinical trials: Reolysin, which is a reovirus, and Onyx-015, which is an adenovirus deletion mutant. The various clinical trials revealed that the therapeutic agents were selective for cancer cells, but therapeutic potency was limited. In general, anti-tumor gene therapy has largely failed to date in patients owing to inefficient delivery of the gene to sufficient numbers of cancer cells locally and systemically. Development of new generation anti-tumor drugs should, therefore, focus on improved anticancer potency, improved efficacy of delivery and improved systemic spread.
Interestingly, proteins derived from several of these viruses, i.e., CAV-derived apoptosis-inducing apoptin, adenovirus early region 4 open reading frame (E4orf4) and parvovirus-H1-derived non-structural protein 1 (NS1), were identified as agents that are able to induce aberrant-cell apoptosis. For example, apoptin was shown to be the main aberrant cell-specific apoptosis-inducing factor of CAV. In addition to these apoptosis-inducing proteins identified in these viruses, new apoptosis-inducing proteins were identified that are not part of viruses' genomes but that are also able to induce cell death specifically in aberrant cells. Examples are human α-lactalbumin made lethal to tumor cells (HAMLET), human cytokines melanoma differentiation-associated gene-7 (mda-7) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).
The ability of these viral proteins apoptin, E4orf4 and NS1 and these non-viral cellular proteins HAMLET, TRAIL and mda-7 to induce apoptosis in aberrant cells renders them with a high potency for beneficial incorporation in anti-tumor therapies.
Parvovirus-H1 NS1 protein induces cell death in glioma cells. The tumor-selective apoptosis-inducing activity of NS1 is related to its interaction with the catalytic subunit of casein kinase II (CKIIα). Formation of NS1-CKIIα complexes points to interference by NS1 with intracellular signaling processes (Noteborn, Eur. J. Pharm., 2009). As a result of the formed NS1-CKIIα complexes, CKIIα-dependent cytoskeletal changes occur followed by apoptosis. Parvovirus-H1 infections induce characteristic changes within the cytoskeleton filaments of tumor cells, which results finally in the degradation of actin fibers and the appearance of so-called actin patches.
Loss of p53 functioning is related to tumor formation and is at the basis of resistance of tumors to various anticancer therapies. The adenovirus-derived protein E4orf4 selectively kills tumor cells independent of p53 (Noteborn, Eur. J. Pharm., 2009). Like parvovirus-H1-derived protein NS1, E4orf4 expression results in deregulation of the cytoskeleton. E4orf4-induced cell death is not dependent on classical caspase pathways, and E4orf4 circumvents Bcl-2 blockage of apoptosis and does not require release of mitochondrial cytochrome c. Seemingly, E4orf4 is able to trigger apoptosis in aberrant cells via an alternative cell death process not present in non-aberrant cells.
Human α-lactalbumin made lethal to tumor cells (HAMLET) is a structural derivative of α-lactalbumin, a main protein of human milk. HAMLET can induce apoptosis in a tumor-selective manner (Noteborn, Eur. J. Pharm., 2009). The precursor of HAMLET is α-lactalbumin, which undergoes structural changes upon binding of oleic acid and subsequent release of calcium ions. HAMLET can specifically kill aberrant cells of skin papillomas, glioblastoma tumors, and bladder cancers by efficient uptake, leaving healthy tissue unaltered. HAMLET acts on the caspase pathways due to stimulated release of cytochrome c from the mitochondria. In the nuclei of tumor cells, HAMLET associates with histones resulting in an irreversible disruption of the chromatin organization. This seems the key event responsible for the tumor-cell killing activity of HAMLET, apart from its ability to activate 20S proteasomes. HAMLET induces tumor-selective apoptosis in a p53-independent manner.
Melanoma differentiation-associated gene-7 (mda-7; interleukin 24), an interleukin-10 family member, induces apoptosis in various cancer cells dependent on caspases (Noteborn, Eur. J. Pharm., 2009). For example, apoptosis-inducing activity of mda-7 upon down-regulation of survival signals such as Bcl-2 and Akt by mda-7 is seen in breast cancer cells when adenoviral-induced mda-7 is used. Also secreted mda-7 exposes anti-tumor cell activity on distant tumor cells. Specificity of mda-7 apoptosis-inducing activity is based on the activation of the FasL/TRAIL pathways. Mda-7 has been proven effective pre-clinically in treatment of subcutaneous ovarian cancer xenografts and lung tumor xenografts (combination therapy), when adenovirus-expressing mda-7 was used. A clinical phase I trial revealed that subsets of tumor cells are resistant to mda-7, leaving substantial room for further improvement of therapies based on proteins bearing apoptosis-inducing activity.
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces both p53-dependent and p53-independent apoptosis in tumor cells (Noteborn, Eur. J. Pharm., 2009). TRAIL activates the extrinsic apoptosis pathway leading to caspase 8 and subsequently amongst other caspase-3 activation. Subsequently, TRAIL-induced apoptosis activates the intrinsic apoptosis pathway. One of the first steps in TRAIL-induced apoptosis is the binding of TRAIL to death receptors DR4 and DR5. TRAIL's apoptosis activity is selective for tumor cells but the diversity of tumor cells susceptible to TRAIL-induced apoptosis is limited. This is perhaps due to the fact that TRAIL signaling also activates NF-κB, which induces anti-apoptotic regulators. In addition or alternatively, TRAIL resistance of several types of tumor cells may be due to the fact that these tumor cells over-express anti-apoptosis protein FLIP or Bcl-2.
The CAV-derived apoptin is a viral protein with apoptosis-inducing activity toward a broad range of human aberrant cell types but not toward normal, non-transformed human diploid cells including primary human hepatocytes and stem cells. A broad variety of tumor cell types is susceptible to apoptin's apoptosis-inducing activity. This apoptin activity can be triggered by induced transformation of cells. These two observations point to regulation of the apoptosis pathway by apoptin during an early stage of the cell transformation process. The specificity of apoptin for tumor cells may be related to its multimeric nature when in its active form, its interaction with chromatin structures in tumor cells, its selective phosphorylation in malignant cells, and its ability to elevate ceramide levels in tumor cells, which is a tumor suppressor activity. This latter activity is indicative for an important role of sphingolipids in apoptin-induced apoptosis. Apoptin induces apoptosis also by acting on and interfering with the cell cycle processes. That is to say, apoptin acts mainly via interaction with the anaphase-promoting complex/cyclosome complex, inducing G2/M cell cycle arrest resulting in p73/PUMA-mediated apoptosis. Cytochrome c release and activation of the central caspase pathways are involved in apoptin-induced cell death. The selectivity of apoptin's apoptosis-inducing activity for tumor cells is p53 independent and, in several tumor cell types, is not sensitive to Bcl-xl and even stimulated by Bcl-2. In noimal cells, apoptin is found located mainly in the cytoplasm. In transformed cells and in malignant cells characterized by metaplasia, hyperplasia or dysplasia, apoptin localizes (also) in the nucleus (Danen-van Oorschot et al., 1997).
Application of apoptin biology has been tested for its efficiency in selectively killing tumor cells in a series of in vitro and in vivo cancer models. Thus far, apoptin has shown a beneficial apoptosis-inducing effect pre-clinically in the context of hepato-carcinoma, breast carcinoma, lung cancer, liver cancer and prostate cancer. Exposing tumor cells to apoptin resulted in a slowdown of tumor growth or even a complete regression of tumors, when delivered to cancer cells intra-tumoral via a non-replicative adenovirus, for the treatment of hepatoma (when part of the Fowl-pox virus genome) (Li et al., Int. J. Cancer, 2006). Beneficial effects of apoptin treatment were also reported for Lewis lung carcinoma, when delivered to the aberrant cells as part of plasmid DNA and for hepato-carcinoma, when applying the Asor-DNA delivery approach. For lung tumors, cervix carcinomas, gastric cancer and hepato-carcinomas, apoptin proved effective when recombinant apoptin was used complexed with a polypeptide for tunneling apoptin into targeted cells, i.e., the protein transfer domain TAT protein of HIV or PTD4. Apoptin was beneficial in the treatment of osteosarcoma and prostate cancer, when combined in combinatorial therapeutic approaches (Olijslagers et al., Basic Clin. Pharmacol. Toxicol., 2007). On the other side, apoptin has been proven to be inactive regarding its apoptosis-inducing activity in normal lymphoid cells, dermal cells, epidermal cells, endothelial cells and smooth muscle cells, providing further insight in the cancer cell specificity of apoptin (Danen-van Oorschot et al. 1997).
Apoptin, comprising 121 amino-acid residues, consists of proline-rich regions, two basic C-terminal clusters K82-R89 and R111-R120 and, over all, contains a high percentage of serine and threonine residues. The two basic clusters comprise the apoptin nuclear localization signal in the apoptin 81-121 amino-acid residues fragment. These clusters Ruin a tumor-selective apoptosis domain, regulated by phosphorylation of threonine residue 108 (additionally, apoptin comprises four serine phosphorylation sites in total). A second tumor-selective apoptosis domain is located at the N-teiininus of apoptin and is a hydrophobic domain, involved in apoptin multimerization (apoptin amino-acid residues 1-69) and comprising interaction sites for other, possibly numerous proteins. Multimerization of apoptin results in protein globules, predominantly spherical in shape, consisting of approximately 30 apoptin molecules each. These homogenous oligomerized apoptin globules have tumor-selective apoptosis-inducing activity. The apoptin is approximately 30 mers and can be soluble in nature, or can be insoluble.
Based on the secondary structure prediction results of five different algorithms, feeding the algorithms with the full-length apoptin sequence 1-121 (SEQ ID NO:3), the apoptin amino-acid sequence 32Glu-Leu46 (e.g., amino acids 32-46 of SEQ ID NO:3) encompasses two predicted beta-strands: 32Glu-Ile-Arg-Ile35 (amino acids 32-35 of SEQ ID NO:3) and 40Ile-Thr-Ile-Thr-Leu-Ser45 (amino acids 40-45 of SEQ ID NO:3), of which the latter is possibly extended with 39Gly and/or with Leu46. Circular dichroism spectropolarimetry experiments with an apoptin-His6 construct indeed revealed that apoptin multimers built up of approximately 30 mers have adopted beta-sheet secondary structure to a small extent. The consensus beta-strands allow for formation of an anti-parallel intra-molecular beta-sheet in apoptin molecules. This beta-sheet encompasses two beta-strands: strand a, residues 32-Glu-Ile-Arg-Ile-35 (amino acids 32-35 of SEQ ID NO:3), and strand b, residues 40-Ile-Thr-Ile-Thr-43 (amino acids 40-43 of SEQ ID NO:3), linked by residues 36-Gly-Ile-Ala-Gly-39 (amino acids 36-39 of SEQ ID NO:3). Amino-acid residues Ile33, Ile35, Ile40 and Ile42 form a hydrophobic face at one side of the intra-molecular beta-sheet; Glu32, Arg34, Thr41 and Thr43 form a charged and hydrophilic opposite face of the same beta-sheet. Thus, hydrophobic side chains of all Ile residues are located at one side of the beta-sheet, with all hydrophilic and charged side chains pointing outward at the opposite side of the anti-parallel beta-sheet. With eight amino acid residues in beta-sheet conformation in apoptin 30-mer globules, in theory, 6.6% beta-sheet content could be determined with a CD measurement. With a hydrophobic face and a charged/hydrophilic face, protein surfaces are formed at apoptin that are accessible for incorporation in an inter-molecular amyloid-like structure build up by, apparently, approximately 30 apoptin molecules. The hydrophobic beta-sheet faces of apoptin molecules will form binding interactions and the hydrophilic/charged beta-sheet faces of apoptin molecules will form binding interactions. It appears that the formation of amyloid-like structure resulting in approximately 30 mers is an intrinsic capacity of apoptin related to its tumor-specific apoptosis-inducing activity in transformed and aberrant cells.
In an earlier application (WO02/079222, Fusion proteins for specific treatment of cancer and auto-immune diseases), a polypeptide complex is disclosed with apoptosis-inducing activity and a viral vector comprising the nucleic acid encoding this polypeptide that achieves the goal of (specifically) killing aberrant cells, e.g., tumor cells, by targeting these cells and, as a result, specifically inducing apoptosis in these tumor cells. It is believed that this eradication of aberrant cells is the result of uptake of the polypeptide or of the viral vector bearing the nucleic acid encoding this polypeptide bearing apoptosis-inducing activity, by both aberrant cells and non-transfoimed healthy cells, followed by selective induction of apoptosis in the aberrant cells only, leaving the healthy cells basically unaltered.